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United States Department of Agriculture

Agricultural Research Service

Title: Cloning and Expression of Porcine Recombinant Soluble Tumor Necrosis Factor Receptor 1

Authors
item Boury, Nancy - IOWA STATE UNIV., AMES
item Bosworth, Brad
item Stabel, Thomas
item Kehrli Jr, Marcus
item Taylor, Michael - IOWA STATE UNIV., AMES

Submitted to: American Journal of Veterinary Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 18, 1998
Publication Date: N/A

Interpretive Summary: Tumor necrosis factor is a substance produced by the white blood cells of animals infected with a variety of common, economically important bacterial agents such as Salmonella and E. coli. While tumor necrosis factor may have a limited role in recovery from disease, it often has negative side effects, such as chronic weight loss. We made a protein which blocks the effects of tumor necrosis factor. This blocking protein will be useful in determining the negative side effects of tumor necrosis factor and their economic impact in pigs with disease. More importantly, this protein will be able to prevent or treat the negative side effects of tumor necrosis factor. Preventing chronic weight loss has the potential to save pork producers millions of dollars a year. Consumers are the eventual beneficiaries of this product because of the availability of economically produced pork.

Technical Abstract: Objective: The objectives of this study were to clone, sequence and express porcine soluble tumor necrosis factor I (sTNFR1) and to determine the bioactivity of recombinant porcine sTNFR1. Procedure: A PCR-based library enrichment technique was used to isolate a fragment of porcine TNFR1. The mature extracellular domain of porcine TNFR1 was subcloned into an expression vector and expressed in Escherichia coli as a FLAG fusion protein. An anti-FLAG affinity column was used to purify the fusion protein. The bioactivity of the purified protein was tested for its ability to inhibit TNF-mediated cytotoxicity in a PK(15) bioassay. Results: A 927-bp fragment of porcine TNFR1 encoding the entire extracellular and transmembrane domains as well as 75 amino acids of the cytoplasmic domain was isolated from a porcine lung cDNA library. The extracellular domain was expressed as a soluble TNFR1 fusion protein with a yield of 120-150 ug per liter of culture. Affinity purified porcine sTNFR1 was able to inhibit TNF-mediated cytotoxicity of porcine PK(15) cells in a dose-dependent manner. Conclusions: Porcine recombinant soluble TNFR1 inhibits TNF bioactivity in vitro. This recombinant protein may be useful in studying the roles of both TNF and TNFR1 in the pathogenesis of infectious diseases in swine.

Last Modified: 7/23/2014
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